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Objectives: A decline in mitochondrial function and increased susceptibility to oxidative stress is a hallmark of ageing. Exercise endogenously generates reactive oxygen species (ROS) in skeletal muscle and promotes mitochondrial remodelling resulting in improved mitochondrial function. It is unclear how exercise induced redox signalling results in alterations in mitochondrial dynamics and morphology. Methods: In this study, a Caenorhabditis elegans model of exercise and ageing was used to determine the mechanistic role of Peroxiredoxin 2 (PRDX-2) in regulating mitochondrial morphology. Mitochondrial morphology was analysed using transgenic reporter strains and transmission electron microscopy, complimented with the analysis of the effects of ageing and exercise on physiological activity. Results: The redox state of PRDX-2 was altered with exercise and ageing, hyperoxidised peroxiredoxins were detected in old worms along with basally elevated intracellular ROS. Exercise generated intracellular ROS and rapid mitochondrial remodelling, which was disrupted with age. The exercise intervention promoted mitochondrial ER contact sites (MERCS) assembly and increased DAF-16/FOXO nuclear localisation. The prdx-2 mutant strain had a disrupted mitochondrial network as evidenced by increased mitochondrial fragmentation. In the prdx-2 mutant strain, exercise did not activate DAF-16/FOXO, mitophagy or increase MERCS assembly. The results demonstrate that exercise generated ROS increased DAF-16/FOXO transcription factor nuclear localisation required for activation of mitochondrial fusion events that were blunted with age. Conclusions: The data demonstrate the critical role of PRDX-2 in orchestrating mitochondrial remodelling in response to a physiological stress by regulating redox dependent DAF-16/FOXO nuclear localisation.

We aimed to study the influence of preventing methemoglobin (metHb) formation, in the roles of peroxiredoxin 2 (Prx2), glutathione peroxidase (GPx) and catalase (CAT) on the erythrocyte antioxidant defense system. We performed in vitro assays using healthy erythrocytes, with and without inhibition of autoxidation of Hb (saturation with carbon monoxide), followed by H 2 O 2 -induced oxidative stress. We assessed the enzyme activities and amounts of CAT, GPx and Prx2 in the red blood cell (RBC) cytosol and membrane and several biomarkers of oxidative stress, such as the reduced and oxidized glutathione levels, thiobarbituric acid reactive substances (TBARS) levels, membrane bound hemoglobin and total antioxidant status. When autoxidation of Hb was inhibited, no significant changes were found for GPx and CAT; Prx2 was observed only in the monomeric form in the cytosol and none bound to the membrane. Blocking the function of Hb as a pseudo-peroxidase does not seem to have an impact on the function of the RBC peroxidases., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Sarcomas are rare and clinically diverse malignancies, and treatment optimization requires the development of suitable biomarkers. In earlier research employing proteomics analysis, we identified peroxiredoxin 2 as a biomarker of osteosarcoma (OS) that can predict response to neoadjuvant chemotherapy and verified its functional significance in the resistance of OS cells to chemotherapeutic drugs. In addition, in gastrointestinal stromal tumor (GIST), we identified pfetin as a prognostic biomarker and validated its prognostic utility in multi-institutional studies by immunohistochemistry. Here, we present an overview of our progress in sarcoma proteomics and discuss future perspectives.

Catalase (CAT), glutathione peroxidase (GPx) and Prx2 (peroxiredoxin 2) are the main antioxidant enzymatic defenses of erythrocytes. They prevent and minimize oxidative injuries in red blood cell (RBC) components, which are continuously exposed to oxidative stress (OS). The crosstalk between CAT, GPx and Prx2 is still not fully disclosed, as well as why these typically cytoplasmic enzymes bind to the RBC membrane. Our aim was to understand the interplay between CAT, GPx and Prx2 in the erythrocyte's cytosol and membrane. Under specific (partial) inhibition of each enzyme and increasing H 2 O 2 -induced OS conditions, we evaluated the enzyme activities and amounts, the binding of CAT, GPx and Prx2 to RBC membrane, and biomarkers of OS, such as the reduced and oxidized glutathione levels, thiobarbituric acid reactive substances (TBARS) levels, membrane bound hemoglobin and total antioxidant status. Our results support the hypothesis that when high levels of H 2 O 2 get within the erythrocyte, CAT is the main player in the antioxidant protection of the cell, while Prx2 and GPx have a less striking role. Moreover, we found that CAT, appears to have more importance in the antioxidant protection of cytoplasm than of the membrane components, since when the activity of CAT is disturbed, GPx and Prx2 are both activated in the cytosol and mobilized to the membrane. In more severe OS conditions, the antioxidant activity of GPx is more significant at the membrane, as we found that GPx moves from the cytosol to the membrane, probably to protect it from lipid peroxidation., Competing Interests: Declaration of competing interest The authors report no declarations of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Hereditary Spherocytosis (HS) is a non-immune hemolytic anemia associated to oxidative stress (OS), namely to the linkage of cytosolic antioxidant enzymes to the erythrocyte membrane. Our aims were to evaluate erythrocyte OS changes and the membrane linkage of peroxiredoxin 2 (Prx2), glutathione peroxidase (GPx) and catalase (CAT) in unsplenectomized (unspl) and splenectomized (spl) HS patients and to search for associations with clinical severity (in unspl HS patients). We studied 114 HS patients (74 unspl and 40 spl) and 30 healthy individuals and we evaluated membrane bound hemoglobin (MBH), membrane lipid-peroxidation (LPO), enzymatic activities of GPx and CAT and the amounts of membrane bound Prx2, GPx and CAT, as well as, clinical and analytical parameters for characterization of HS. We found that unspl HS patients showed clear signs of anemia and in spl HS, a correction to this anemia was observed; the latter patients presented higher levels of OS biomarkers, namely, MBH and LPO. CAT was detected in the membrane of all individuals (control and HS groups), while GPx and Prx2 were only present in HS patients; moreover, their linkage to the membrane (in HS) appears to be cumulative since membrane bound peroxidases amount was higher as the number of peroxidases detected increased. MBH increased with the number/amount of membrane bound peroxidases, however LPO levels remained similar. In conclusion, our data suggest that the binding of these typically cytosolic peroxidases to erythrocyte membrane may be part of a mechanism of membrane protection to maintain its integrity by possibly regulating LPO., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Peroxiredoxins (Prxs) detoxify hydrogen peroxide (H 2 O 2 ), peroxynitrite, and various organic hydroperoxides. However, the differential oxidative status of Prxs reacted with each peroxide remains unclear. In the present study, we focused on the oxidative alteration of Prxs and demonstrated that, in human red blood cells (RBCs), peroxiredoxin 2 (Prx2) is readily reactive with H 2 O 2 , forming disulfide dimers, but was not easily hyperoxidized. In contrast, Prx2 was highly sensitive to the relatively hydrophobic oxidants, such as tert-butyl hydroperoxide (t-BHP) and cumene hydroperoxide. These peroxides hyperoxidized Prx2 into oxidatively damaged forms in RBCs. The t-BHP treatment formed hyperoxidized Prx2 in a dose-dependent manner. When organic hydroperoxide-treated RBC lysates were subjected to reverse-phase high performance liquid chromatography, two peaks derived from hyperoxidized Prx2 appeared along with the decrease of that corresponding to native Prx2. Liquid chromatography-tandem mass spectrometry analysis clearly showed that hyperoxidation to sulfonic acid (-SO 3 H) at Cys-51 residue was more advanced in a newfound hyperoxidized Prx2 compared to another hydrophobic hyperoxidized form previously identified. These results indicate that irreversible hyperoxidation of the Prx2 monomer in RBCs was easily caused by organic hydroperoxide but not H 2 O 2 . Thus, it is important to detect the hyperoxidation of Prx2 into sulfinic or sulfonic acid derivates of Cys-51 because hyperoxidized Prx2 is a potential marker of oxidative injury caused by organic hydroperoxides in human RBCs., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Insulin signaling is essential for regulating glucose homeostasis. Numerous studies have demonstrated that reactive oxygen species (ROS) affect insulin signaling, and low ROS levels can act as a signal to regulate cellular function. Peroxiredoxins (Prxs) are highly abundant and widely expressed antioxidant enzymes. However, it is unclear whether antioxidant enzymes, such as Prx2, mediate insulin signaling. The aim of our study was to investigate the influence of Prx2 deficiency on insulin signaling. Our western blot results showed that Prx2 deficiency enhanced insulin signaling and increased oxidation of protein tyrosine phosphatase 1B (PTP1B) and phosphatase and tensin homologue (PTEN) in mouse embryonic fibroblasts (MEFs) treated with insulin. In addition, we assessed ROS levels with a Cytosol-HyPer H 2 O 2 sensor. As a result, increased ROS levels and Akt activation were decreased by N-acetyl-cysteine (Nac), which acted as an antioxidant in Prx2-deficient MEFs. Body weight measurements and glucose tolerance test (GTT) revealed significant body weight reduction and increase in glucose clearance in Prx2 -/- mice fed a high-fat diet. Interestingly, glucose transporter type 4 (GLUT4) was significantly higher in Prx2 -/- mice than in wild-type mice according to western blotting results. Western blotting also revealed that Akt phosphorylation was higher in Prx2 -/- MEFs and muscle tissue than in wild-type. Together, our findings indicate that increased ROS due to Prx2 deficiency promotes insulin sensitivity and glucose clearance in skeletal muscles by increasing protein tyrosine phosphatase (PTPs) oxidation. These results provide novel insights into the fundamental mechanisms of insulin signaling induced by Prx2 deficiency and suggest that ROS-based therapeutic strategies can be used to suppress insulin resistance., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Peroxiredoxins (Prxs) are a family of thiol peroxidases, which have been suggested to serve as biomarkers for diseases caused by oxidative stress. In this study, we established a high performance liquid chromatography (HPLC) method for quantifying the amount of Prx2 in red blood cells (RBCs). RBC proteins were separated using HPLC, and a single peak was detected that matched that produced by recombinant Prx2. Under improved conditions, the calibration curve for Prx2 (reduced form) was linear over the range of 0.5-20.0μg with a correlation coefficient of 0.999. The minimum detectable level of the recombinant Prx2 was 0.2μg, with a signal-to-noise ratio of 3 per 20μl of injection volume. SDS-PAGE and mass spectrometric analysis showed that the proteins comprising the peak were almost exclusively Prx2. Further high-resolution analysis using nanoLC-MS/MS demonstrated that the oxidation sensitive, Cys-51 was carbamidomethylated by iodoacetamide-alkylation during in-gel digestion but was not modified with sulfinic acid (-SO2H) or sulfonic acid (-SO3H). These results indicated that the separated Prx2 was the reduced form and not the hyperoxidized form. These basic experiments allowed us to determine the relative amounts of native Prx2 in RBCs taken from healthy subjects. The average levels of Prx2 in male and female subjects were 7.28ng/mg and 8.29ng/mg, respectively, and no significant difference was observed between the sexes. Therefore, the HPLC method with UV detection described herein offers a convenient method to quantitatively determine the levels of reduced form of Prx2 and its oxidative decrease in human RBCs., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Circadian clock is regulated by a transcriptional/translational feedback loop (TTFL) lasting ∼24 h. Circadian oscillation of peroxiredoxins (PRDX1-6) redox status has been shown in mature erythrocytes. We have recently reported that nuclear levels of PRDX2 are circadian regulated in the HaCaT keratinocytes. In this study, we addressed whether PRDX2 translocation could influence the TTFL. A reporter HaCaT cell line stably expressing the luciferase gene under control of Bmal1 promoter was lentivirally transduced either with an empty vector (EV), a vector carrying a myc-tagged wild type PRDX2 (PRDX2-Myc) or the same gene with a nuclear localization sequence (PRDX2-MycNuc). PRDX2 overexpressing cells were protected from H2O2-induced oxidative stress. The amplitude of the Bmal1 promoter activity was significantly dampened in PRDX2-MycNuc versus EV cells when synchronized either by dexamethasone treatment or temperature cycles. Clock synchronization was not affected in PRDX2 silenced cells. N-acetyl cysteine or melatonin treatments, significantly dampened the Bmal1 promoter activity suggesting that sustained scavenging of ROS impairs clock synchronization. Noteworthy, H2O2 treatment rescued proper oscillation of the clock in synchronized PRDX2-MycNuc HaCaT cells. Since the histone deacetylase Sirtuin 1 (Sirt1) modulates clock gene expression amplitude, the effect of Sirt1 activator resveratrol or Sirt1 inhibitor nicotinamide were also investigated. Interestingly, NAM enhanced the molecular clock synchronization in PRDX2-MycNuc cells. Our findings demonstrate that PRDX2 regulates the TTFL oscillation by finely tuning the cellular redox status of the nucleus likely influencing the deacetilase activity of SIRT1 enzyme., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Circadian rhythms are highly conserved time tracking systems regulating important biological processes at both systemic and cellular levels. The present study was aimed to identify proteins and biological functions circadian regulated in human keratinocytes. HaCaT keratinocytes were entrained by temperature cycles, and a proteomic study was performed on cell fractions isolated under free running conditions at constant temperature. Bioinformatics analysis revealed that molecular clock entrainment was associated with changes in molecular components regulating cell proliferation, energy metabolism, transcription, translation and redox balance. Nuclear levels of the antioxidant enzyme Peroxiredoxin 2 (PRDX2) were found to oscillate rhythmically over two entire 24h long cycles. Donwregulation of PRDX2 resulted in upregulation of the mitochondrion-specific Peroxiredoxin 3 (PRDX3), all other members of the Peroxiredoxin family remained unaltered. Furthermore, PRDX2 knockdown increased intracellular levels of reactive oxygen species (ROS) and impaired cell cycle progression and proliferation. HaCaT cells transduced with a scramble shRNA were used as control. Our work is the first to show that nuclear levels of PRDX2 display circadian oscillation participating in the regulation of human keratinocytes redox balance., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

We attempted to identify biomarkers that would predict responsiveness of osteosarcoma (OS) to induction chemotherapy. Tumor tissues obtained by open biopsy before induction chemotherapy were investigated. On the basis of histological observations at the time of surgery and the Huvos grading system, 7 patients were classified as good responders and the other 6 as poor responders. Protein expression profiling was performed by two-dimensional difference gel electrophoresis. Among 3494 protein spots observed, the intensity of 33 spots was found to differ significantly between the two patient groups. The proteins for these 33 protein spots were identified by mass spectrometry. The higher expression of peroxiredoxin 2 (PRDX2) in poor responders was confirmed by Western blotting. Gene silencing assay demonstrated that reduced expression of PRDX2 was associated with increased sensitivity of OS cells to chemotherapeutic drugs such as methotrexate, doxorubicin and cisplatin. Moreover, siRNA-induced silencing of PRDX2 resulted in a decrease of cell proliferation, invasion and migration. These findings indicated that PRDX2 would be a candidate biomarker of response to induction chemotherapy. Measurement of PRDX2 in open biopsy samples before treatment may contribute to risk stratification therapy for OS., Biological Significance: The response of osteosarcoma patients to induction chemotherapy is critical because the prognosis of responders is quite favorable, whereas that of non-responders is poor. Although there are many therapeutic options for osteosarcoma, no parameter for predicting the response to induction chemotherapy has been available. We conducted a proteomics study aimed at developing a biomarker that would predict the response of osteosarcoma to induction chemotherapy. Using open biopsy samples obtained before chemotherapy, we conducted 2D-DIGE with our originally devised large-format electrophoresis apparatus and identified peroxiredoxin 2 (PRDX2) as a novel predictive biomarker. The diagnostic performance of PRDX2 was confirmed by ROC analysis, and its functional properties were investigated in a series of in vitro functional assays. Our findings indicate the possible application of PRDX2 as a predictive biomarker in patients with osteosarcoma., (© 2013.)

Lung cancer (LC) and chronic obstructive pulmonary disease (COPD) commonly coexist in smokers, and the presence of COPD increases the risk of developing LC. The aim of this study was to identify distinct proteomic profiles able to discriminate these two pathological entities. Protein content was assessed in the bronchoalveolar lavage (BAL) of 60 patients classified in four groups: COPD, COPD and LC, LC without COPD, and control with neither COPD nor LC. Proteins were separated into spots by bidimensional polyacrylamide gel electrophoresis (2D-PAGE) and examined by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF/TOF). A total of 40 proteins were differentially expressed in the LC and/or COPD groups as compared with the control group. Distinct protein profiles were identified and validated for each pathological entity (LC and COPD). The main networks involved were related to inflammatory signalling, free radical scavenging and oxidative stress response, and glycolysis and gluconeogenesis pathways. The most relevant signalling link between LC and COPD was through the NF-κB pathway. In conclusion, the protein profiles identified contribute to elucidate the underlying pathogenic pathways of both diseases, and provide new tools of potential use as biomarkers for the early diagnosis of LC., Biological Significance: Sequence coverage. The protein sequence coverage (95%) was estimated for specific proteins by the percentage of matching amino acids from the identified peptides having confidence greater than or equal to 95% divided by the total number of amino acids in the sequence. Ingenuity Pathways Analysis. Mapping of our proteins onto biological pathways and disease networks demonstrated that 22 proteins were linked to inflammatory signalling (p-value: 1.35 10(-08)-1.42 10(-02)), 15 proteins were associated with free radical scavenging and oxidative stress response (p-value: 4.93 10(-11)-1.27 10(-02)), and 9 proteins were related with glycolysis and gluconeogenesis pathways (p-value: 7.39 10(-09)-1.58 10(-02))., (Copyright © 2013 Elsevier B.V. All rights reserved.)